Native Taylor/Non‐Taylor Dispersion–Mass Spectrometry (TNT‐MS) Allows Rapid Protein Desalting and Multiplexed, Label‐Free Ligand Screening

Native mass spectrometry (MS) is an important technique in structural biology and drug discovery, due to its ability to study non-covalent assemblies in the gas phase. Drawbacks include the incompatibility of electrospray ionization (ESI) with non-volatile salts and the risk of protein signal suppression by small molecules. Overcoming these often requires offline buffer exchange and/or parallel sample preparation to other methods, reducing the adoption and throughput of native MS. Here, we exploit the dynamics of analytes flowing through an open tubular capillary to keep molecules with a small hydrodynamic radius (e.g., salts) inside a Taylor dispersion regime while pushing larger species (e.g., proteins) into a non-Taylor regime. As such, larger species elute earlier, and are effectively buffer exchanged within the capillary in seconds. In addition to desalting of proteins injected in biologically relevant buffers we demonstrate separation of unbound small molecules from protein-ligand complexes, enabling multiplexed ligand screening. Finally, we investigated the dependence of the critical flow rate for non-Taylor behavior on protein size, enabling limited size-based separation of proteins. Taylor/non-Taylor dispersion mass spectrometry (TNT-MS) was implemented using an unmodified liquid chromatography - mass spectrometry (LC-MS) system operated without a chromatographic column and coupled to an autosampler, which allowed significant automation.